Interferons are in a special class of antiviral proteins secreted in minute amounts from mammalian cells upon induction with viruses, double-stranded RNAs, immunotoxins, mitogenes, etc. There are two main types of interferon: type I represented by the interferons α (lymphocyte interferon) and β (fibroblasts interferon) and type II (or immune interferon) represented by the interferon γ (IFN). The interferon family has been extremely well characterized in the prior art (Haus, L., Archivum Immunologiae et Therapiae Experimentalis, 2000, 48, 95-100.).
The interferon (IFN) system is one of the major mechanisms involved in human immunity. Interferons (IFNs) are a family of related cytokines that mediate a range of diverse functions including antiviral, antiproliferative, antitumor, and immunomodulatory activities. Its disregulation may result in a greater tendency to infectious diseases and to the development of cancer. Genes of interferon system proteins are often located at the sites of breakpoints of the structural chromosome aberrations in cancer.
IFN's are pH stable interferons produced by leukocytes and fibroblasts in response to viral infections. Both alpha and beta IFN belong to class I interferons. The IFNα gene family (about 26 genes, including pseudogenes) and the IFNβ gene are located at band 21 of the chromosome 9 short arms (9p21) the latter more distally than the former29. IFNα and IFNβ are intronless genes originating from a common ancestor gene. (Jaramillo et al., (1995): The interferon system. A review with emphasis on the role of PKR in growth control. Cancer Invest., 13, 327-338; MCK KU@ SICK V. A. (1998): Mendelian inheritance in man. A catalog of human genes genetic disorders. 12th ed. The Johns Hopkins Univ. Press, Baltimore-London.). The human interferon gene family is fully described in this and other references cited throughout the entirety of this application. More specifically, the art has described in detail a number of IFN genes. These genes are well characterized and described in the art. Furthermore, a study of Annu Rev Biochem. 1998; 67:227-64, reveals a number of interferon genes and how cells respond to interferons. These publications are hereby fully incorporated by reference. Furthermore, Henco et al., in J Mol Biol. 1985 Sep. 20; 185(2):227-60, isolated and characterized DNA segments containing IFN-alpha-related sequences from human lambda and cosmid clone banks. They described six linkage groups comprising 18 distinct IFN-alpha-related loci, and report the nucleotide sequences of nine chromosomal IFN-alpha-genes with intact reading frames, as well as of five pseudogenes. Still a further reference which describes a number of the interferon genes is Archivum Immunologiae et Therapiae Experimentalis, 2000, 48, 95-100P L ISSN 0004-069X, The Genes of Interferons and Interferon-Related Factors: Localization and Relationships with Chromosome Aberrations in Cancer. Still, another paper, Biopolymers. 2000; 55(4):254-87, provides a review of the history of the alpha related IFN. The human interferon gene cluster on the short arm of chromosome 9 comprises 26 genes the functional members of which are separated by highly efficient scaffold.
Recombinant IFNα2b is being used for the treatment of Hepatitis B and C for several types of cancer. However, the IFNα2b drugs that are being marketed are produced through an E. coli expression system and due to necessary in vitro processing and purification, the average cost of treatment is $26,000 per year. Patients are normally injected with the drugs, Intron®A and PEG-Intron™, resulting in severe side effects which have been linked to route of administration. Because oral delivery of natural human IFNα2b has been shown to elicit a systemic immune response without the negative side effects, it is desirable to create an analogue to natural human IFNα2b that is suitable for oral administration to mammals.
The microbial species used to produce the IFNα2b is marketed under the names PEG-Intron™ and Intron®A is E. coli. Prokaryotic expression systems have many advantages as production systems for heterologous proteins. They can be cultured in large quantities inexpensively and in a short time by standard methods of fermentation (Walsh, 1998). In addition, E. coli has been well characterized, with over 40 recombinant proteins produced in E. coli already approved for general medical use (Walsh, 2000).
However, many eukaryotic proteins cannot be expressed in prokaryotic hosts because their mRNAs contain introns that need to be removed in order for correct translation and E. coli is unable to process these transcripts (Glick and Pasternak, 1998). The IFNαs are unusual for eukaryotic proteins in that they contain no introns, and so processing is not necessary. Although numerous IFN α subtypes have been expressed in E. coli, special techniques that add to the cost of the drug have to be employed to produce the mature, biologically active interferon. Prokaryotic systems cannot form disulfide bonds when IFNα is produced intracellularly and consequently it cannot fold properly (Thatcher and Panayotatos, 1986). As a result, the IFNαs, such as IFNα2b, aggregate to form inclusions bodies that need to be solubilized (Swaminathan and Khanna, 1999). Additional downstream processing steps include purification and formation of proper disulfide bonds (Walsh, 1998). Besides E. coli, low levels of IFNα2 have been expressed in silkworm using a baculovirus vector (Maeda et al., 1985) and into a phage vector (Slocombe et al., 1982).
For several viruses and cancers, the only treatment approved by the FDA is injections of IFNα2b. However, the treatment has many side effects and only 20% of patients who need treatment can actually afford to buy the drug (Harris-Stuart and Penny, 1997). Consequently, alternative means of producing IFNα2 have been explored.
Although bacterial and fungal systems are the most predominant systems for commercial production of recombinant proteins, they have several important drawbacks when producing proteins from eukaryotes. Proteins that require disulfide bonds or glycosylation are not well suited for expression in microorganisms (Glick and Pasternak, 1998). A recombinant protein can be toxic to the microorganism, form inclusion bodies, or be degraded by proteases (Kusnadi et al., 1997). Transgenic plants are potentially one of the most economical systems for large-scale production of recombinant proteins for industrial and pharmaceutical uses (Walmsley and Arntzen, 2000).
Unique to plants is the ability to regenerate whole plants from cells or tissues. This totipotency has many practical benefits: for example, plants propagated by seed can be cultured in vitro to yield thousands of identical plants (Bhojwani, 1990). In particular, tobacco is the easiest plant to genetically engineer and is widely used to test suitability of plant-based systems for bioproduction of recombinant proteins. Tobacco is an excellent biomass producer (in excess of 40 tons leaf fresh weight/acre based on multiple mowings per season) and a prolific seed producer (up to one million seeds produced per plant), thus hastening the time in which a product can be scaled up and brought to market (Cramer et al., 1998). In general, plant systems are more economical than industrial facilities using fermentation or bioreactor systems and the technology is already available for harvesting and processing plants and plant products on a large scale (Daniell et al., 2001a). Plant-derived products are less likely to be contaminated with human pathogenic microorganisms than those derived from animal cells because plants don't act as hosts for human infectious agents (Giddings et al., 2000).
Recombinant proteins expressed in plant cells are naturally protected from degradation when taken orally (Kong et al., 2001). Oral delivery is highly desirable for drug treatment (Gomez-Orellan and Paton, 1998). Oral administration of natural human IFNα has proven to be therapeutically useful in the treatment of various infectious diseases and low doses of recombinant IFNαs were shown to be effective as well (Tompkins, 1999).
The genetic information of plants is distributed among three cellular compartments: the nucleus, the mitochondria, and the plastids and each of these carries its own genome and expresses heritable traits (Bogorad, 2000). Transformation of the plant nucleus is routine in many species and there are a variety of techniques for delivering foreign. DNA to the plant nuclear genome (Hager and Bock, 2000). However, recombinant protein expression in plants by nuclear transformation have been low, with most levels much less than the 1% of total soluble protein that is needed for commercial feasibility if the protein must be purified (Daniell et al., 2002). For example, only 0.000017% of transgenic tobacco leaves was IFN (Elderbaum et al., 1992). Also, negligible amounts of IFNα was produced in nuclear transformation of rice (Zhu et al., 1994). In addition, with nuclear expression, the foreign protein levels vary in transgenic lines because the foreign gene is inserted randomly into different locations (Bogorad, 2000). Other factors that lower expression levels are the gene silencing and position effects so often observed in nuclear transgenic plants (Daniell and Dhingra, 2002).
The plastids of plants are an attractive target for genetic engineering. Plant plastids (chloroplasts, amyloplasts, elaioplasts, etioplasts, chromoplasts, etc.) are the major biosynthetic centers that, in addition to photosynthesis, are responsible for production of industrially important compounds such as amino acids, complex carbohydrates, fatty acids, and pigments. Plastids are derived from a common precursor known as a proplastid and thus the plastids present in a given plant species all have the same genetic content. In general, plant cells contain 500-10,000 copies of a small 120-160 kilobase circular plastid genome, each molecule of which has a large (approximately 25 kb) inverted repeat. Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest which can result in very high levels of foreign gene expression.
The modern chloroplast of plants has retained a largely prokaryotic system of gene organization and expression, with the eukaryotic nuclear genome exerting significant regulatory control (Hager and Bock, 2000). Signaling pathways have evolved to coordinate gene expression between the chloroplast and the nuclear-cytosolic compartments during chloroplast development and in response to environmental factors such as light (Zerges, 2000). Illuminated chloroplasts possess extraordinarily high rates of transcription and translation that is tissue-specific due to regulation via untranslated regions of chloroplast-encoded mRNAs. Although communication between the chloroplast and the nucleus exist, these membrane-separated genetic systems have their own distinct environmental milieu containing different proteins, proteases and mechanisms of action. Unique features of the photosynthetic plastid enable genetic engineering of the chloroplast to overcome major limitations of plant nuclear transformation technology.
One major concern with the genetic modification (GM) of plants is the possibility of the escape of foreign genes through pollen dispersal from transgenic plants to sexually compatible weedy relatives or to pathogenic microbes in the soil (Daniell, 2002). Such gene transfers could potentially result in the emergence of “superweeds” able to resist certain herbicides thereby undermining the benefits of GM crops (Daniell, 2002). However, genes in the chloroplasts of higher plants are generally transmitted only by the maternal parent, which means that chloroplast genes are not present in the pollen (Bogorad, 2000). Therefore, a foreign gene introduced by genetic engineering of the chloroplast genome could not transfer to genetically compatible weeds. This uniparental or maternal inheritance provides the gene containment necessary for keeping foreign genes sequestered in target plants and preventing gene flow among crops and weeds (Daniell, 2002).
Another remarkable feature of the plastid genome is its high ploidy level: a single tobacco leaf cell may contain as many as 100 chloroplasts, each harboring approximately 100 identical copies of the plastid genome, resulting in an extraordinarily high ploidy degree of up to 10,000 plastid genomes per cell (Bogorad, 2000). Because of the very high ploidy level of the plastid genome, very high expression levels can be achieved. For example, the Bacillus thuringiensis (Bt) Cry2Aa2 protein accumulated as cuboidal crystals in transgenic chloroplasts and reached a level of 45.3% of the tsp in mature leaves (De Cosa et al., 2001).
For transformation of chloroplasts in plants, particle bombardment is used to introduce transgenes into leaf chloroplasts and stable transformation requires that 10,000 chloroplast copies be uniformly converted (Bock and Hagemann, 2000). Securing genetically stable lines of plants with transgenic chloroplast requires every chloroplast to carry the inserted gene (Bogorad, 2000). This homoplasmic state is achieved through amplification and sorting of transgenic chloroplasts with the elimination of the wild-type copies on selective medium (Maliga, 1993). The integration of cloned plastid DNA into the plastid genome occurs through site-specific homologous recombination in plants such as in tobacco N. tabacum and excludes the foreign vector DNA (Kavanagh et al., 1999). In contrast, nuclear transformation experiments in higher plants frequently suffer from epigenetic gene-silencing mechanisms resulting in inconsistent and unstable gene expression or complete loss of transgenic activity (Hager and Bock, 2000). The nuclear genome has mechanisms to effectively inactivate genes when regulatory sequences are inserted in a repetitive pattern and this occurs because integration of transgenes into the nuclear genome is random and through non-homologous recombination (Daniell and Dhingra, 2002). Random integrations of transgenes also means that the final location of the inserted gene may be in a region of the nuclear genome that is not highly transcribed. As a consequence, nuclear expression levels vary in different transgenic lines and these differences are due to the inserted gene's random position in the nuclear genome. Neither gene silencing nor position effects have been observed in genetically engineered chloroplasts may be in region of the nuclear genome that is not highly transcribed. As a consequence, nuclear expression levels vary in different transgenic lines and these differences are due to the inserted gene's random position in the nuclear genome. Neither gene silencing nor position effects have been observed in genetically engineered chloroplasts (Daniell and Dhingra, 2002). Another major advantage of chloroplast engineering is the expression of multiple transgenes as operons due to efficient translation of polycistronic messenger RNAs (De Cosa et al., 2001). Genetic engineering has now moved from introducing single gene traits to coding for complete metabolic pathways, bacterial operons, and biopharmaceuticals that require assembly of complex multi-subunit proteins (Daniell, 2002).
Disulfide bonds are common to many extracellular proteins because they stabilize the native conformation by lowering the entropy of the unfolded form (Abkevich and Shakhnovich, 2000). Most proteins need to be folded correctly for the protein to function properly and remain in solution. Eukaryotic secretory proteins are normally routed through the endoplasmic reticulum where disulfide bond formation occurs. Experiments show that chloroplasts have the machinery needed to fold complex eukaryotic secretory proteins in the soluble chloroplast stroma compartment. The activities of several chloroplast enzymes involved in the anabolic processes of carbon assimilation are enhanced or triggered by light through a signaling system called the ferredoxin-thioredoxin system (Ruelland and Miginiac, Maslow, 1999). Two correct disulfide bonds were formed in the tobacco chloroplast expression of human somatotropin. In another study, binding assays confirmed that chloroplast-synthesized cholera toxin of Vibrio cholera (CTB) bound intestinal receptors indicating that correct folding and disulfide bond formation had occurred (Daniell et al., 2001). The light signal sensed by chlorophyll is transferred via the photosynthetic electron flow to proteins called thioredoxins, which are very efficient in thio-disulfide interchanges with various protein disulfides (Ruelland and Miginiac-Maslow, 1999). Another mechanism for the simple, reversible activation of genes that regulate expression in the chloroplast is the Protein Disulfide Isomerase (PDI) system composed of chloroplast polyadenylate-binding proteins that specifically bind to the 5′UTR of the psbA mRNA and are modulated by redox status through PDI (Kim and Mayfield, 1997). The ability of chloroplasts to form disulfide bonds and properly fold foreign proteins eliminates a major part of the costly downstream processing.
Expression of functional human somatotropin in transgenic tobacco chloroplasts established that chloroplasts are capable of proper folding of human proteins with disulphide bonds. The ability to express multiple genes in a single transformation event (Daniell and Dhingra, 2002; De Casa et al., 2001), accumulation of exceptionally large quantities of foreign proteins (De Casa et al., 2001), successful engineering of tomato chromoplasts for high level transgene expression in fruits (Ruf et al., 2001, or carrots (Kumar et al., 2003), coupled to hyper-expression of vaccine antigens (Daniell et al., 2001b), and the use of plant derived antibiotic free selectable markers (Daniell et al., 2001c), augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most. The term “edible vaccine” or “oral delivery” as used herein refers to a substance which may be given orally which will elicit a protective immunogenic response in a mammal.
Good recombinant systems are still not available for many human proteins that are expensive to purify or highly susceptible to proteolytic degradation. It is known that traditional purification of biopharmaceuticals proteins using columns accounts for 30% of the production cost and 70% of the set up cost (Petrides et al., 1995). Proteolytic degradation is another serious concern for industrial bioprocessing. The increasing production of proteins in heterologous hosts through the use of recombinant DNA technology has brought this problem into focus; heterologous proteins appear to be more prone to proteolysis (Enfors, 1992). Recombinant proteins are often regarded by a cell as foreign and therefore degraded much faster than most endogenous proteins (Rozkov et al., 2000). Proteolytic stability of recombinant proteins is a significant factor influencing the final yield. In view of these limitations, the Applicant has developed a more efficient method for producing a recombinant biopharmaceutical protein, such as IFNα2b production, which may be used as a model system to enrich or purify biopharmaceutical proteins from transgenic plants, which are highly susceptible to proteolytic degradation.
To date no one has successfully transformed the plastid genome with IFN to create a delivery system that is easily administered and that stimulates both arms of the immune system without the severe side effects experienced by patients in current IFNα2b treatments. In addition, until the Applicant's discovery, production vehicles (E. coli, nuclear plant genomes, etc. . . . ) have failed to provide a cost effective and functional IFN, which can be orally administered without the side effects, i.e., human pathogens that are associated with the current production vehicles. In view of these limitations the Applicant developed a system for the expression of interferon, such as IFNα2b, via the chloroplast genome in order to provide a feasible means of overproducing this increasingly useful therapeutic drug as well as addressing current concerns with the present methods of delivery and production. Also incorporated by reference into this application is the utility application, based off of U.S. Provisional Application No. 60/393,651, and filed simultaneously with this application. Still another application, PCT/US02/41503, filed on Dec. 26, 2002, is also incorporated by reference into this application. These applications describe in detail, somatic embryogenosis for the construction of edible vaccines.